Through the NIAID-sponsored Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF), we have identified numerous anti-Mycobacterium tuberculosis (M. tb) lead compounds that are structurally similar to purine bases and nucleosides. The goal of this grant proposal is to understand the metabolism of these agents in M. tb and human cells in order to identify the basis for their selective activity. These studies should lead to a thorough understanding of the substrate characteristics of key M. tb purine salvage enzymes. Much is known about the substrate requirements of human enzymes involved in purine metabolism, because of the considerable effort to develop antitumor nucleoside analogs. However, very little is known about the substrate characteristics of purine metabolizing enzymes in M. tb. The proposed studies should remedy this deficiency, and the information gained will be useful in the rational design and development of new agents based on metabolic differences in purine metabolism between humans and M. tb. In our preliminary data we have identified a metabolic difference between M. tb and human purine metabolism that could be involved in the selective activation in M. tb of certain adenosine analogs. However, further work is necessary to completely characterize the mechanism of action of these and other agents. The proposed specific aims to accomplish these goals are: (1) characterization of the metabolism and mechanism of action of adenosine analogs in intact M. tb; (2) isolation and characterization of purine salvage enzymes from M. tb and human sources; (3) design and synthesis of adenosine analogs selective for M. tb; (4) design and synthesis of a combinatorial library of 2 and 6-substituted purities; (5) preliminary biochemical evaluation of compounds synthesized in aim 4; and (6) evaluation of compounds for anti-M. tb activity and toxicity.